(1) Summary of Invention
The present invention relates to isolated and purified DNA encoding a carbohydrate binding protein or lectin, designated as BJ38, which is a segment of chromosomal DNA of Bradyrhizobium japonicum. The DNA is used to provide the lectin, as a probe and as a basis for producing super nodulating strains of Rhizobium.
(2) Description of Related Art
(2) Background and Rationale
Rhizobia encompass three genera of gram negative bacteria: Rhizobium, Bradyrhizobium and Azorhizobium (Brewin, N. J., Ann. Rev. Cell Biol. 7:191-226 (1991)). Each member of the three classes of rhizobia can nodulate a specific legume: R. leguminosarum bv. viciae nodulates pea and vetch; R. leguminosarum bv. trifolii (hereafter referred to as R. trifolii) nodulates clover; and B. japonicum nodulates soybean. This host specificity is observed throughout the nodulation process, including the early stages before infection initiation. It is most likely determined by multiple levels of interactions between components derived from both partners of the symbiosis (Erewin, N. J., Ann. Rev. Cell Biol. 7:191-226 (1991); Sanchez, F., et al., Ann. Rev. Plant Physiol. Mol. Biol. 42:507-528 (1991); and Fisher, R. F., et al., Nature 357:655:660 (1992)).
First, there are diffusible signals from the plant to the bacteria. Rhizobia are chemotactic towards specific flavonoid compounds released by the legume roots (Brewin, N. J., Ann. Rev. Cell Biol. 7:191-226 (1991); Sanchez, F., et al., Ann. Rev. Plant Physiol. Mol. Biol. 42:507-528 (1991); Fisher, R. F., et al., Nature 357:655:660 (1992); Maxwell, C. A., et al., Plant Physiol. 93:1552-1558 (1990)). The flavonoids induce transcription of an important set of nodulation (nod) genes in rhizobia. This process is mediated by nodD, the only nod gene constitutively expressed (Long, S. R., Cell 56:203-214 (1989)). NodD proteins from different species of rhizobia recognize different flavonoids preferentially and these activate the transcription of the other nod genes. Thus, this molecular recognition constitutes an important first level determinant of host-Rhizobium specificity (Brewin, N. J., Ann. Rev. Cell Biol. 7:191-226 (1991); Sanchez, F., et al., Ann. Rev. Plant Physiol. Mol. Biol. 42:507-528 (1991); Fisher, R. F., et al., Nature 357:655:660 (1992)).
Second, there are diffusible signals from the bacterium to the plant. The induction of bacterial nod genes results in the synthesis and secretion of Nod factors. Host specificity here is determined by species-specific chemical modification of the signal molecules. Nod factors secreted by R. meliloti, whose preferred host is alfalfa, and R. leguminosarum bv. viciae, which nodulates pea or vetch, are modified .beta.-1,4-linked oligomers of N-acetyl-D-glucosamine (Lerouge, P., et al., Nature 344:781-784 (1990); and Spaink, H. P., et al., Nature 354:125-130 (1991)). The sulfate group of the R. meliloti factor is an important host specificity determinant (Ehrhardt, D. W., et al., Science 256: 998-1000 (1992) ), whereas the O-acetyl group and the nature of the fatty acyl substituent affect the biological activity of the R. leguminosarum bv. viciae factors (Spaink, H. P., et al., Nature 354:125-130 (1991)). The responses of plant cells to the Nod factors include membrane potential depolarization (Ehrhardt D. W., et al., Science 256:998-1000 (1992)), expression of genes specific for stages of infection and nodule organogenesis (the nodulins) (Horvath, B., et al., Plant J. 4:727-733 (1993)), and morphogenetic alterations such as curling of the root hairs (Truchet, G, et al., Nature 351:670-673 (1991)). The synthesis of the Nod factors is mediated by certain nod genes, which are under the transcriptional regulation of nodD protein (Brewin, N. J., Ann. Rev. Cell Biol. 7:191-226 (1991)).
The third level in determining host specificity occurs at the attachment of the rhizobia to the root surface (Wang, J. L., et al., Trends Glycosci. Glycotech. 5:331-342 (1994); Ho, S. C., et al., In: Lectin Reviews, (Kilpatrick, D. C., et al.,, eds.) Vol. 1, pp. 171-181, Sigma, St. Louis, MO, USA (1991); and Dazzo, F. B., J. Supramol. Struct. Cell Biochem. 16:29-41 (1981)). When the bacterial cells are inoculated on the seedling roots, they rapidly clump at the tips of the root hair cells. Over the course of several hours, the bacteria attach in a polar (end-to-end) fashion along the sides of the root hair. Within a few days, a marked curling of the root hair tip is induced and occasionally, bacteria entrapped within the curl penetrate the root hair cell wall to form a tubular structure called the "infection thread". The invading bacteria induce proliferation of cortical cells in the root, which eventually emerge as a nodule. These nodules contain bacteria that can reduce atmospheric nitrogen into ammonia, which is assimilated by the host plant.
Soybeans have vaulted into second place in value among all U.S. crops. They are surpassed only by corn. They outrank even corn as the single largest U.S. agricultural crop export. Soybeans are mainly used in two main products, meal and oil. They provide high quality human and animal foods as well as industrial products, including salad oil, high protein foods, like tofu, and a substitute for petroleum in industrial uses. The success of research directed agriculture is enlightening. Thanks to past research, national average yields have climbed from 23 bushels per acre in 1960 to about 37 at the present time. Researchers have even hit as many as 118 bushels per acre in maximum yield studies.
The attachment of B. japonicum to soybean root has been thoroughly analyzed (Wang, J. L., et al., Trends Glycosci. Glycotech. 5:331-342 (1993)). A galactose/lactose-specific bacterial lectin, namely BJ38 (mol. wt. 38,000) (Ho, S. C., et al., J. Cell Biol. 111:1639-1643 (1990) ) was identified and immunolocalized at one pole of the bacteria coincident with the point of attachment when the bacteria attached to the soybean root. This suggests a role for BJ38 in host recognition. Two binding deficient mutants showed no surface expression of BJ38 and significantly deficient in modulation activity (Ho, S. C., et al., J. Cell Biol. 111:1631-1638 (1990); and Ho, S. C., et al., Plant Journal, 5:873-884 (1994)). This established the importance of BJ38 for attachment and its involvement in nodulation. Further support of this idea comes from the strong correlation between three activities along the young root hair zone: bacterial attachment, activation of nod genes, and the susceptibility of the emergent root hair for nodulation.
In the Bradyrhizobium japonicum-soybean interaction, the attachment of bacteria to the host-legume is galactose (Gal)-specific (Vesper, S. J., et al., Symbiosis 1:139-162 (1985); Ho, S.-C., et al., J. Cell biol. 111:1631-1638 (1990); and Smith, G. B., et al., Can. J. Microbiol. 39:245-251 (1992)), suggesting the involvement of a carbohydrate-binding protein. Consistent with this observation, the isolation of the carbohydrate binding protein, BJ38, from B. japonicum was documented. BJ38 exhibits a saccharide specificity similar to that of bacterial adhesion to soybean roots (Ho, S.-C., et al., J. Cell biol. 111:1639-1643 (1990)). In addition the lectin has also been immunolocalized at the attachment site of bacterial attachment to soybean cells (Loh, J. T., et al., Proc. Natl. Acad. Sci. 90:3033-3037 (1993)). Purified BJ38 binds to soybean roots at sites coincident with B. japonicum attachment: namely, at the emergent root hair zones (Ho, S.-C., et al., Plant J. 5:873-884 (1994)). These regions had previously been demonstrated by Bauer and coworkers (Bhuvaneswari, T. V., et al., Plant Physiol. 66:1027-1031 (1980); and Calvert, H. E., et al., Can. J. Bot. 62:2375-2384 (1984)) to be the most susceptible to initiation of infection by B. japonicum. It has been reported that the expression of BJ38 can be induced by both lactose (Lac) and the isoflavonoid genistein (Loh, J. T., et al., Glycoconjugate J., 11:363-370 (1994)). As genistein is a potent inducer of the nod genes of B. japonicum, this latter result suggests the possibility that BJ38 may be a member of the nod gene family.
The nod genes comprise a set of key bacterial elements in the infection process and are transcribed in response to specific flavonoid compounds secreted from the host-plant (Peters, N. K., et al., Science 233:917-1008 (1986); Zaat, S. A. J., et al., Plant Physiol. 86:1298-1303 (1988); and Kosslak, R. M., et al., Proc. Natl. Acad. Sci. USA 84:7428-7432 (1987)). This requires the presence of the nodd gene product (Mulligan, J. T., et al., Proc. Natl. Acad. Sci. USA 82:6609-6613 (1985); Rossen, L., et al., EMBO J 4:3369-3373 (1985); and Banfalvi, Z., et al., Mol. Gen. Genet. 214:420-424 (1988)), which, in association with the appropriate flavonoids, binds to the nod box promoter sequence preceding the nod genes and activates the transcription of these genes (Fisher, R. F., et al., Genes Dev. 2:282-293 (1988); and Hong, G. G., et al., Nucleic Acids Res. 15:9677-9690 (1987)). In B. japonicum, two copies of nodd, nodD.sub.1 and noD.sub.2, have been identified (Gottfert, M., et al., Mol. Plant-Microbe Interact. 5:257-265 (1992)). Of these two nodD genes, only nodD.sub.1 is both inducible by is of lavonoids and necessary for nod gene induction.
The patent art relating to Rhizobium is substantial and is exemplified by U.S. Pat. Nos. 4,713,330 to McLouchlin, 4,782,022 to Puhler et al, 4,803,165 to Appelbaum, 4,818,696 to Appelbaum et al, 4,863,866 to Zablotowicz, et al, 4,966,847 to Stacey et al, 4,983,519 to Stacey, et al, 5,001,061 to Rolfe et al, 5,008,194 to Rolfe et al, 5,023,180 to Appelbaum et al,5,045,461 to Scott, 5,059,533 to Watson et al,5,059,534 to Appelbaum, 5,077,209 to O'Gara, 5,124,260 to Hauke et al, 5,137,816 to Rolfe et al,5,141,745 to Rolfe et al,5,183,759 to Triplett, 5,229,113 to Kosslak et al, and European Patent Appln. 0 289 947A1.
There is a need to isolate super nodulating strains of B. japonicum for use as inoculants to enhance soybean crop yield based on their superiority in binding and infecting soybean root at the sites for nodulation and on the superiority in competitiveness against other indigenous Rhizobium strains which are inferior in nitrogen-fixing efficiency. There is also a need to isolate the DNA encoding BJ38.